Use of modified pyrimidine compounds to promote stem cell migration and proliferation

ABSTRACT

This invention provides cells and methods for stimulating proliferation and migration of endogenous and exogenous mammalian stem cells in vivo and in vitro. The invention provides reagents and methods for efficiently proliferating mammalian stem cells in an animal in need thereof and producing stem cells that can be re-introduced into an animal in need thereof to alleviate neurological and corporal disorders.

[0001] This application is related to U.S. Provisional PatentApplication Serial No. 60/348,473, filed Jan. 14, 2002, and Serial No.60/357,783, filed Feb. 19, 2002, and Serial No. 60/376,257, filed Apr.29, 2002, and Serial No. 60/381,138, filed May 8, 2002, and Serial No.60/404,361, filed Aug. 19, 2002, and Serial No. 60/430,381, filed Dec.2, 2002, the disclosures of each of which are expressly incorporated byreference herein.

[0002] This invention was made with support from the U.S. Governmentthrough the National Institutes of Health, grant no. R03-AG 19874. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to methods for stimulating proliferationand migration of mammalian stem cells in vivo and in vitro and cellsproduced by those methods. In particular, the invention providesreagents and methods for efficiently proliferating stem cells in ananimal in need thereof and producing stem cells that can bere-introduced into an animal in need thereof to alleviate neurologicaland corporal disorders.

[0005] 2. Background of the Related Art

[0006] Stem cells are often defined as self-renewing and multipotent,with the ability to generate diverse types of differentiated cells. Assuch, they show promise in the treatment of neurological and corporaldisorders (also referred to as neurological and corporal “deficits”), orany loss or diminishment of tissue function due to age, disease, traumaor other factor. However, such treatments have faced significant hurdlesthat have yet to be substantially overcome.

[0007] NSCs and Neurological Deficits

[0008] Because an important focus of stem cell replacement therapies hasbeen neurological disorders, neural stem cells, and particularly fetalneural stem cells, have been a major research target. During developmentof the central nervous system (CNS), multipotent neural stem cells(MNSCs), also known as multipotent precursor cells (MPCs), ortissue-specific neural stem cells (NSCs), proliferate, giving rise totransiently dividing progenitor cells that eventually differentiate intothe cell types that compose the adult brain, including neurons,astrocytes and oligodendrocytes. NSCs have been isolated from severalmammalian species, including mice, rats, pigs and humans. See, e.g.,International Application, Publication Nos. WO 93/01275, WO 94/09119, WO94/10292, WO 94/16718 and Cattaneo et al., 1996, Mol. Brain Res. 42:161-66. NSCs from the embryonic and adult rodent central nervous system(CNS) have been isolated and further propagated in vitro in a variety ofculture systems. See, e.g., Frolichsthal-Schoeller et al., 1999,NeuroReport 10: 345-351; Doetsch et al., 1999, Cell 97: 703-716. NSCsfrom the human fetal brain have been cultured using serum-free mediumsupplemented with epidermal growth factor (EGF) and/or basic fibroblastgrowth factor (bFGF). See, e.g., Svendsen et al., 1998, J. Neurosci.Meth. 85: 141-152; Carpenter et al., 1999, Exp. Neurol. 158: 265-278.NSCs cultured utilizing these serum-free, mitogen-supplemented methodsgenerally form substantially undifferentiated, clustered aggregates.Upon removal of the mitogen(s) and provision of a substrate, theseneural stem cells differentiate into neurons, astrocytes andoligodendrocytes.

[0009] While the synaptic connections involved in neural circuits arecontinuously altered throughout the life of the individual, due tosynaptic plasticity and cell death, neurogenesis (the generation of newneurons) was thought to be complete early in the postnatal period. Thediscovery of MNSCs in the adult brain (see, e.g., Alvarez-Buylla et al.,1997, J. Neurobiol. 33: 585-601; Gould et al., 1999, Science 286:548-552) has significantly changed the theory on neurogenesis, as thepresence of MNSCs in the adult brain suggests that regeneration ofneurons can occur throughout life. Nevertheless, age, physical andbiological trauma or neurodegenerative disease-associated loss of brainfunction, herein referred to as a “neurological deficit,” can faroutweigh any potential restorative effects due to endogenousneurogenesis. As a result, up-regulated or stimulated proliferation ofendogenous MNSCs as well as transplantation of MNSCs are potentiallyvaluable treatments for those suffering from the loss of, or loss ofappropriate, brain function due to age, physical and biological traumaor neurodegenerative disease (i.e., a neurological deficit). No suchtreatments are known in the art.

[0010] Due to the advancing average age of the population, andconcomitantly increased incidence of neurological deficit thataccompanies advancing age, treatment of neurodegenerative diseases hasbecome a major concern. Such diseases, including Alzheimer's disease,Huntington's chorea and Parkinson's disease, have been linked toneuronal degeneration at specific locations in the brain leading to theinability of the brain region to synthesize and—releaseneurotransmitters that are vital to neuronal signaling.

[0011] Neurodegeneration also encompasses many conditions and diseases,age-related or not, that result in neuronal loss. These conditionsinclude CNS trauma, such as ischemia (stroke) and epilepsy, as well asdiseases that result in neuronal loss, including amyotrophic lateralsclerosis and cerebral palsy.

[0012] Many such neurological deficits are localized to particularregions of the brain. Degeneration in a brain region known as the basalganglia can lead to diseases with varied and different cognitive andmotor symptoms, depending on the exact location of the lesion. The basalganglia consists of many separate regions, including the striatum (whichconsists of the caudate and putamen), the globus pallidus, thesubstantia nigra, substantia innominata, ventral pallidum, nucleusbasalis of Meynert, ventral tegmental area and the subthalamic nucleus.

[0013] Degeneration in the basal ganglia can lead to motor deficits. Forexample, Huntington's chorea is associated with degeneration of neuronsin the striatum, which leads to involuntary jerking movements.Degeneration of a small region called the subthalamic nucleus isassociated with violent flinging movements of the extremities in acondition called ballismus, while degeneration in the putamen and globuspallidus are associated with a condition of slow writhing movements orathetosis. In Parkinson's disease, degeneration is seen in another areaof the basal ganglia, the substantia nigra par compacta. This areanormally sends dopaminergic connections to the dorsal striatum, whichare important in regulating movement. Therapy for Parkinson's diseasehas centered upon restoring dopaminergic activity to this circuit.

[0014] Alzheimer's disease patients exhibit a profound cellulardegeneration of the forebrain and cerebral cortex. Further, a localizedarea of the basal ganglia, the nucleus basalis of Meynert, appears to beselectively degenerated. This nucleus normally sends cholinergicprojections to the cerebral cortex that are thought to participate incognitive functions including memory.

[0015] The objective of most CNS therapies is to regain the particularchemical function or enzymatic activity lost due to cellulardegeneration. Administration of pharmaceutical compositions has been themain treatment for CNS dysfunction though this type of treatment hascomplications, including the limited ability to transport drugs acrossthe blood-brain barrier, and drug-tolerance acquired by patients to whomthese drugs are administered for long periods.

[0016] Transplantation of multipotent stem cells may avert the need notonly for constant drug administration, but also for complicated drugdelivery systems necessitated by the blood-brain barrier. In practice,however, significant limitations have been found in this technique aswell. First, cells used for transplantation that carry cell surfacemolecules of a differentiated cell from a donor can induce an immunereaction in the recipient, a problem that is exacerbated by the physicaldamage caused by injection of cells directly into the affected area ofthe brain. In addition, the neural stem cells must be at a developmentalstage where they are able to form normal neural connections withneighboring cells.

[0017] For these reasons, initial studies on neurotransplantationcentered on the use of fetal cells.

[0018] Mammalian fetal brain tissue has proven to have reasonablesurvival characteristics upon immediate transplantation. Increasedsurvival capability of fetal neurons is thought to be due to the reducedsusceptibility of fetal neurons to anoxia compared to adult neurons. Anadditional factor favoring survival of fetal cells is their lack of cellsurface markers, whose presence may lead to rejection of grafted tissuefrom adults. However, although the brain is considered animmunologically privileged site, some rejection of even fetal tissue canoccur. Therefore, the ability to use heterologous fetal tissue islimited by tissue rejection and the resulting need for immunosuppressantdrug administration.

[0019] The use of large quantities of aborted fetal tissue presentsother difficulties as well. Fetal CNS tissue is composed of more thanone cell type, and thus is not a well-defined tissue source. Inaddition, it may be unlikely that an adequate and constant supply offetal tissue would be available for transplantation. For example, in thetreatment of MPTP-induced Parkinsonism, tissue from as many as 6 to 8fetuses can be required for successful implantation into the brain of asingle patient. There is also the added problem of the potential forcontamination during fetal tissue preparation. Since this tissue mayalready be infected with a bacteria or virus, expensive diagnostictesting is required for each fetus used. Even comprehensive diagnostictesting might not uncover all infected tissue. For example, there can beno guarantee that a sample is HIV-free, because antibodies to the virusare generally not present until several weeks after infection.

[0020] In addition to fetal tissue, there are other potential sources oftissue for neurotransplantation, including cell lines and geneticallyengineered cell types, but both sources have serious limitations. Celllines are immortalized cells that are derived, inter alia, bytransformation of normal cells with an oncogene or by the culturing ofcells in vitro with altered growth characteristics. Moreover, adverseimmune response potential, the use of retroviruses to immortalize cells,the potential for the reversion of these cells to an amitotic state, andthe lack of response by these cells to normal growth-inhibiting signalsmake such cell lines sub-optimal for widespread use.

[0021] Another approach to neurotransplantation involves the use ofgenetically engineered cell types or gene therapy. However, there stillexists a risk of inducing an immune reaction with these cells. Inaddition, retrovirus mediated transfer may result in other cellularabnormalities. Also, cell lines produced by retrovirus-mediated genetransfer have been shown to gradually inactivate their transferred genesfollowing transplantation and further may also not achieve normalneuronal connections with the host tissue. Currently availabletransplantation approaches suffer from significant drawbacks. Theinability in the prior art of the transplant to fully integrate into thehost tissue, and the lack of availability of suitable cells in unlimitedamounts from a reliable source for grafting are significant limitationsof neurotransplantation. Studies utilizing intra-tissue injection ofdissociated and partially differentiated NSCs have shown little promise(see, e.g., Benninger et al., 2000, Brain Pathol. 10: 330-341; Blakemoreet al. 2000, Cell Transplant 9: 289-294; Rosser et al., 2000, Eur. J.Neurosci. 12: 2405-2413; Rubio et al., 2000, Mol. Cell Neurosci. 16:1-13). The results have generally been poor because, among manyconsiderations, the dissociation of clusters of NSCs is known to causeimmediate senescence of NSCs and increase the vulnerability of NSCs inculture. See, e.g., Svendsen et al., 1998, J. Neurosci. Meth. 85:141-152. Further, regardless of adverse immune responses provoked byforeign tissue being introduced into the brain, the trauma caused by thephysical introduction of cells directly into the damaged area can inducethe recruitment of immune cells by the host that can eliminate thetransplanted cells. Thus, significant problems with the use of NSCs toameliorate neurological deficits remain. As described herein,neurological deficits also include non-brain tissues such as, forexample, the eye and spinal cord.

[0022] A “corporal deficit” is a disorder caused by a wide variety ofdiseases and injuries, resulting in trauma, malfunction, degeneration orloss of muscle such as, for example, cardiac muscle due to myocardialinfarction. Other examples include malfunction, degeneration or loss ofother cells and tissues apart from those discussed in the neurologicaldeficit section above such as, for example, internal organs. Forexample, liver function can be adversely affected by, among otherthings, disease (e.g., cirrhosis or hepatitis), trauma or age. Theproblems described above in using NSCs to remedy neurological deficitsof the brain also apply to neurological deficits in other tissues, suchas the eye, and corporal deficits.

[0023] There exists a need in the art for improved methods forincreasing the number of multipotent cells in an animal and therebyincreasing the reservoir of remedial capacity conferred by multipotentstem cells in tissues. There exists a need to stimulate proliferation,migration or both proliferation and migration of endogenous andexogenously introduced mammalian multipotent stem cells in vivo as wellas mammalian multipotent stem cells in vitro. There exists a need forcells stimulated to proliferate, migrate or both proliferate andmigrate, as well as pharmaceutical compositions for treating aneurological deficit or corporal deficit comprising such stimulatedcells. Further, there exists a need in the art for methods ofadministration of such cells stimulated to proliferate, migrate or bothproliferate and migrate and pharmaceutical compositions thereof. Stillfurther, there exists a need for methods for treating an animal having aneurological or corporal deficit.

SUMMARY OF THE INVENTION

[0024] This invention provides methods for stimulating proliferation,migration or both proliferation and migration of mammalian stem cells invivo and in vitro and cells produced by those methods. In particular,the invention provides reagents and methods for efficientlyproliferating stem cells in an animal in need thereof and producing stemcells that can be re-introduced into an animal in need thereof toalleviate neurological disorders.

[0025] In a first aspect, the invention provides a method of stimulatingproliferation, migration or both proliferation and migration ofendogenous and exogenous mammalian stem cells in vivo. In oneembodiment, the method comprises the step of introducing to a mammal aneffective amount of a pyrimidine derivative of:

[0026] where R₁ to R₈ independently represent a hydrogen atom, a loweralkyl group, CH₃OCH₂CH₂—, CH₂CONH₂, —COCH₃, —COC₂H₅ or —CH₂OCOC₂H₅; and

[0027] X is ═NH, ═N—CH₃, ═N—C₂H₅, ═N-ph, ═N—COOC₂H₅, ═N—SO₂CH₃, ═CH₂,═CHCH₃, ═CHC₂H₅, —O— or —S— in which ph stands for a phenyl group; or apharmaceutically acceptable salt thereof.

[0028] In another aspect, the invention provides a method of stimulatingproliferation, migration or both proliferation and migration ofexogenous mammalian stem cells in vivo to a mammal that has had moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells administered thereto. In one embodiment, themethod comprises the step of introducing to a mammal an effective amountof the pyrimidine derivative of formulae (I) or (II) above or apharmaceutically acceptable salt thereof.

[0029] In another aspect, the invention provides a method of stimulatingproliferation, migration or both proliferation and migration ofendogenous mammalian stem cells in vitro. In one embodiment, the methodcomprises the step of contacting a mammalian stem cell with an effectiveamount of the pyrimidine derivative of formulae (I) or (II) above or apharmaceutically acceptable salt thereof.

[0030] In another aspect, the invention provides a method for treatingan animal with a neurological or corporal deficit. In one embodiment,the method comprises the step of administering an effective amount ofthe pyrimidine derivative of formulae (I) or (II) above, or apharmaceutically acceptable salt thereof, wherein the endogenous stemcell population is stimulated to proliferate and migrate to an area oftissue damage, differentiate in a tissue-specific manner and function ina manner that reduces the neurological or corporal deficit. In certainembodiments the inventive methods further comprise the step ofadministering more developmentally potent cells, wherein the moredevelopmentally potent cells are stimulated to proliferate and migrateto an area of tissue damage, differentiate in a tissue-specific mannerand function in a manner that reduces the neurological or corporaldeficit. In related embodiments, the inventive method comprisesadministering autologous or non-autologous stem cells, wherein theautologous or non-autologous stem cells are stimulated to proliferateand migrate to an area of tissue damage, differentiate in atissue-specific manner and function in a manner that reduces theneurological or corporal deficit. In further related embodiments, themore developmentally potent cells or the autologous stem cells or thenon-autologous stem cells administered with the pyrimidine derivativeform a cluster of two or more cells. In further related embodiments, themore developmentally potent cells or the autologous stem cells or thenon-autologous stem cells are derived from a tissue or tissue-specificstem cell. In other embodiments, the stem cell is a hematopoietic stemcell, a neural stem cell, an epithelial stem cell, an epidermal stemcell, a retinal stem cell, an adipose stem cell or a mesenchymal stemcell, any of which can be obtained from any tissue containing stem cellsincluding but not limited to zygote, blastocyst, embryo, fetus, infantjuvenile or adult, and optionally, a human species of any of thepreceding embodiments, whether naturally occurring or engineered. Incertain embodiments, the cluster of two or more of the moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells comprises less than about 50 percentredifferentiated cells, or more preferably less than about 25 percentredifferentiated cells, or even more preferably less than about 10percent redifferentiated cells, or even more preferably less than about5 percent redifferentiated cells, or even more preferably less thanabout 1 percent redifferentiated cells. In related embodiments, the moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells, in the form of a cluster of two or more cellsin other related embodiments, are administered by injecting the moredevelopmentally potent cells with a syringe, inserting the moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells with a catheter or surgically implanting thesaid cells. In other, further related embodiments, the moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells are injected with a syringe, inserted with acatheter or surgically implanted either to a body cavity that isfluidly-connected to the area of neurological or corporal deficit or tothe area of neurological or corporal deficit. In embodiments relating toneurological or corporal deficits, the neurological deficit isoptionally caused by a neurodegenerative disease, a traumatic injury, aneurotoxic injury, ischemia, a developmental disorder, a disorderaffecting vision, an injury or disease of the spinal cord, ademyelinating disease, an autoimmune disease, an infection, or aninflammatory disease and the corporal deficit is optionally caused bycorporal disease, disorder, injury, trauma, malfunction, degeneration orloss

[0031] In certain embodiments the pyrimidine derivative of formula (I)is MS-818, or 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H)pyrrolo[2,3-d]pyrimidine maleate (the C₄H₄O₄ maleate salt), as disclosedin U.S. Pat. No. 4,959,368, incorporated by reference herein. In certainin vivo embodiments, the pyrimidine derivatives of formulae (I) and (II)is administered at a concentration of between about 0.01 mg/kg/day to 50mg/kg/day, more preferably between about 0.1 mg/kg/day to 10 mg/kg/day,even more preferably between about 1 mg/kg/day to 5 mg/kg/day, and evenmore preferably about 3 mg/kg/day. In these embodiments, the pyrimidinederivatives of formulae (I) and (II) is administered for between about 1and 60 days, or more preferably between about 1 and 30 days, or morepreferably between about 1 and 15 days, or even more preferably betweenabout 1 and 10 days, or more preferably between about 2 and 7 days, oreven more preferably about 5 days. In certain others of theseembodiments, the methods further comprise the step of administering agrowth factor. In certain embodiments, the growth factor comprisesfibroblast growth factor, epidermal growth factor or a combinationthereof.

[0032] In certain in vitro embodiments, the stem cell culture iscontacted with the pyrimidine derivative of formulae (I) or (II) in aneffective amount, or a concentration of between about 50 nM to 1 mM, ormore preferably between about 500 nM to 500 μM, or even more preferablybetween about 1 μM to 100 μM, or more preferably between about 5 μM to75 μM and even more preferably about 50 μM. In these embodiments, thestem cell culture is contacted with pyrimidine derivatives of formulae(I) and (II) for an effective period, or between about 1 and 60 days, ormore preferably between about 1 and 30 days, or more preferably betweenabout 1 and 15 days, or even more preferably between about 1 and 10days, or more preferably between about 2 and 7 days, or even morepreferably about 5 days. In certain others of these embodiments, themethods further comprise the step of contacting the cell culture with agrowth factor. In certain embodiments, the growth factor comprisesfibroblast growth factor, epidermal growth factor or a combinationthereof. In certain others of these embodiments, the methods furthercomprise contacting the stem cell culture with heparin.

[0033] In another aspect, the invention provides cells stimulated forproliferation, migration or both proliferation and migration producedaccording to the methods of the invention. In another aspect, theinvention provides a pharmaceutical composition for treating aneurological or corporal deficit comprising the cells stimulated forproliferation, migration or both proliferation and migration producedaccording to the methods of the invention. In certain embodiments, thepharmaceutical composition further comprises a pharmaceuticallyacceptable carrier.

[0034] Thus, the invention advantageously provides methods ofstimulating proliferation and migration of mammalian stem cells in vivoand in vitro, cells produced by those methods, pharmaceuticalcompositions to treat neurological and corporal deficits, and methods ofadministering the cells and pharmaceutical compositions of theinvention.

[0035] Specific embodiments of the present invention will become evidentfrom the following more detailed description of certain preferredembodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee, pursuant to 37 C.F.R. §1.84.

[0037]FIG. 1 shows the effect of transplantation of MNSC according tothe methods of co-owned and co-pending U.S. patent application, entitled“Novel Mammalian Multipotent Stem Cells and Compositions, Methods ofPreparation and Methods of Administration Thereof,” (Ser. No. ______filed Jan. 14, 2003) on memory score in the Morris water maze test. (a)Individual memory score before and after transplantation showsimprovement in the majority of the animals. Blue: Aged memory impairedanimals, Green: Aged memory unimpaired animals, Red: Matured animals.(b) Mean of memory score in each animal group before (n arrow stripedbar) and after (black bar) SC transplantation shows a significantimprovement in aged memory impaired and young animals. The animals thatreceived vehicle injection do not show significant difference in memoryscore between before (wide striped bar) and after (hatched) theinjection. The methods of the instant invention can act to increase thenumber of such exogenously transplanted cells in vivo, as well asenhance their number while being treated according to the methods ofsaid co-owned and co-pending application. Further, the methods of theinstant invention can increase the abundance of the endogenous NSCpopulation.

[0038]FIG. 2 shows typical fluorescent immunohistochemicalphotomicrographs of aged rat brain 30 days after transplantation ofMNSCs of the co-owned and co-pending U.S. patent application referencedabove. bIII-tubulin and GFAP immunoreactivity were used as markers forneuron and glia, respectively. (a) MNSCs of the co-owned and co-pendingU.S. patent application migrated into the cortex and differentiated intoneurons as indicated by the bIII-tubulin positive cells (green), whichhave morphologies typical of pyramidal cells in layer IV and V of theparietal cortex. Apical dendrites were pointed towards to the edge ofthe cortex. Since the NSCs were pre-treated with BrdU, the transplantedcells have BrdU positive nuclei (red). Contrarily, the host cell'snuclei are counter stained with DAPI (blue). M any cells having BrdUpositive nuclei are observed with bIII-tubulin immunoreactivity in layerII and without bIII-tubulin immunoreactivity in layer III. (b, c) Highermagnification of the parietal cortex in cortex layer IV: all thebIII-tubulin immunoreactive (green) positive cells show BrdU (red)positive nuclei while many other host cell's nuclei are stained withonly DAPI (blue). (d) MNSCs according to said co-owned and co-pendingU.S. patent application migrated into the hippocampus and differentiatedinto bIII-tubulin positive cells (green), in CA1 pyramidal cell layer.These bIII-tubulin positive cells have BrdU positive nuclei (red),indicating that these cells originated from transplanted cells. Incontrast, host cell nuclei counter stained with DAPI (blue) are notbIII-tubulin positive. (e) In the dentate gyrus many fibers werebIII-tubulin positive in addition to the bIII-tubulin positive cells(green) and GFAP positive sells (red). (f) bIII-tubulin positive cells(green) and GFAP positive cells (red) were found in layer IV and layerIII, respectively. Such a layer of astrocytes was not observed in normalrats without NSC transplantation. Again, the methods of the instantinvention can act to increase the number of such exogenouslytransplanted, BrdU-treated cells in vivo, as well as enhance theirnumber while being treated according to the methods of the co-owned andco-pending application. The methods of the instant invention can alsoincrease the abundance of the endogenous NSC population.

[0039]FIG. 3 shows the effects of MS-818 on endogenous neural stem cellpopulations in the brain. (a) Typical immunohistochemistry (x200) usingBrdU (brown, marker for proliferating cells) in a control aged ratcerebral cortex without MS-818 treatment. (b) Typicalimmunohistochemistry (x200) using BrdU in an aged rat cerebral cortexwith MS-818 treatment (3 mg/kg/day, i.p. for 5 days). The number of BrdUpositive cells is significantly increased after MS-818 treatment. (c)Typical immunohistochemistry (x200) using BrdU (brown, marker forproliferating cells) in a control aged rat SVZ without MS-818 treatment.(d) Typical immunohistochemistry (x200) using BrdU in an aged rat SVZwith MS-818 treatment (3 mg/kg/day, i.p. for 5 days). The number of BrdUpositive cells is increased after MS-818 treatment. (e) Quantitativeanalysis of the effects of MS-818 on a number of BrdU-positive cells inthe cortex (a, b). There was a 7-fold increase in stem cell populationafter MS-818 treatment.

[0040]FIG. 4 shows the effects of MS-818 on endogenous retinal stem cellpopulations. (a) Typical immunohistochemistry (x400) using BrdU (red,marker for proliferating cells) in control rat retina without MS-818treatment. (b) Typical immunohistochemistry (x400) using BrdU in ratretina with intraocular administration of MS-818 (10 μg/20 μl). Thenumber of BrdU-positive cells is clearly increased after the treatment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0041] This invention provides methods for stimulating proliferation,migration or proliferation and migration of endogenous and exogenousmammalian stem cells in vivo. The invention also provides methods forstimulating proliferation, migration or proliferation and migration ofmammalian stem cells in vitro. The method further provides cellsproduced by the aforementioned methods. More generally, the inventionprovides reagents and methods for efficiently proliferating mammalianstem cells in an animal in need thereof and producing stem cells thatcan be re-introduced into an animal in need thereof to alleviateneurological and corporal disorders.

[0042] As used herein, the terms “multipotent neural stem cells(MNSCs),” “neural stem cells (NSCs),” and “multipotent precursor cells(MPCs)” refer to undifferentiated, multipotent cells of the CNS. Suchterms are commonly used in the scientific literature. MNSCs candifferentiate into tissue-specific cell types, for example astrocytes,oligodendrocytes, and neurons when transplanted in the brain. Themultipotent cells of the invention are distinguished from natural NSCsby their stimulation for proliferation, migration or both proliferationand migration due to treatment by the methods of the invention.

[0043] As used herein, a “less developmentally potent cell” is a cellthat is capable of limited multi-lineage differentiation or capable ofsingle-lineage, tissue-specific differentiation, for example, anuntreated mesenchymal stem cell can differentiate into, inter alia,osteocytes and chrondrocytes, i.e., cells of mesenchymal lineage but hasonly limited ability to differentiate into cells of other lineages(e.g., neural lineage.).

[0044] As used herein, a “more developmentally potent cell” is a cellthat is readily capable of differentiating into a greater variety ofcell types than its corresponding less developmentally potent cell. Forexample, a mesenchymal stem cell can readily differentiate intoosteocytes and chrondrocytes but has only limited ability todifferentiate into neural or retinal lineage cells (i.e., it is a lessdevelopmentally potent cell in this context). Mesenchymal stem cellstreated according to the methods of the above-referenced co-owned andco-pending U.S. patent application become more developmentally potentbecause they can readily differentiate into, for example,mesenchymal-lineage and neural-lineage cell types; the plasticity of thecells is increased when treated according to the methods of theinvention.

[0045] “More developmentally potent cell” and “less developmentallypotent cell” as used herein are fully disclosed and claimed in co-ownedand co-pending U.S. patent application entitled “Novel MammalianMultipotent Stem Cells and Compositions, Methods of Preparation andMethods of Administration Thereof,” Ser. No. ______, filed Jan. 14,2003, or “App. 1.”

[0046] As used herein, “multipotent stem cells” or “MSCs” refer to thecells prepared according to the methods disclosed herein and in co-ownedand co-pending U.S. patent application entitled “Novel MammalianMultipotent Stem Cells and Compositions, Methods of Preparation andMethods of Administration Thereof,” Ser. No. ______, filed Jan. 14,2003, or “App. 1” and co-owned and co-pending U.S. patent applicationentitled “Novel Mammalian Multipotent Neural Stem Cells andCompositions, Methods of Preparation and Methods of AdministrationThereof,” Ser. No. ______, filed Jan. 14, 2003, or “App. 2.” Eachapplication is incorporated herein by reference in their entirety.

[0047] As used herein, the term “cluster” refers to a group of two ormore non-terminally differentiated cells. A cluster can comprise theprogeny of a single multipotent stem cell or small cluster of primarycells.

[0048] As used herein, the terms “effective amount” and “therapeuticallyeffective amount” each refer to the amount of reagent used to support orproduce the desired activity. In the case of the cells stimulated forproliferation, migration or both proliferation and migration preparedand delivered according to the invention, an effective amount is anamount necessary to support or produce an observable level of one ormore biological activities of MSC as set forth herein. Regardingpyrimidine derivatives, an effective amount can be between about 0.01mg/kg/day to 50 mg/kg/day, more preferably between about 0.1 mg/kg/dayto 10 mg/kg/day, even more preferably between about 1 mg/kg/day to 5mg/kg/day, and even more preferably about 3 mg/kg/day.

[0049] An “effective period” as used herein refers to the time periodnecessary for the reagents and cells of the invention to accomplishtheir specified activities. For example, cells of the invention can becontacted with a pyrimidine derivative for an effective period to makethem more developmentally potent. An effective period for contact with apyrimidine derivatives can be, for example, between about 1 and 60 days,or more preferably between about 1 and 30 days, or more preferablybetween about 1 and 15 days, or even more preferably between about 1 and10 days, or more preferably between about 2 and 7 days, or even morepreferably about 5 days.

[0050] The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers to one ormore formulation materials suitable for accomplishing or enhancing thesuccessful delivery of the pharmaceutical composition of stimulated stemcells prepared and delivered according to the invention.

[0051] As disclosed in further detail herein, the inventive methodsprovide for introducing pyrimidine derivatives of formulae (I) or (II),

[0052] where R₁ to R₈ independently represent a hydrogen atom, a loweralkyl group, CH₃OCH₂CH₂—, CH₂CONH₂, —COCH₃, —COC₂H₅ or —CH₂OCOC₂H₅; andX is ═NH, ═N—CH₃, ═N—C₂H₅, ═N-ph, ═N—COOC₂H₅, ═N—SO₂CH₃, ═CH₂, ═CHCH₃,═CHC₂H₅, —O— or —S— in which ph stands for a phenyl group; or apharmaceutically acceptable salt thereof, to a mammal in an amounteffective to stimulate proliferation, migration or both proliferationand migration of endogenous multipotent stem cells in vivo. Endogenousmultipotent stem cells can be of varied origin, inter alia, stem cellsof hematopoietic, neural, mesenchymal, epithelial, epidermal, adiposeand retinal origin, and administration of the pyrimidine derivatives canbe localized to a particular tissue.

[0053] The invention also provides methods for introducing pyrimidinederivatives, or pharmaceutically suitable salts thereof, to a mammal inan amount effective to stimulate proliferation, migration or bothproliferation and migration, in vivo, of exogenous multipotent stemcells introduced to the mammal before, after or concurrently with thepyrimidine derivative. Further, a rest period between the introductionof the pyrimidine derivative and the multipotent stem cells can beimplemented as necessary to minimize any trauma caused by theiradministration. The exogenously introduced multipotent stem cells can beprepared according to the methods described in App. 1 or App. 2, and asset forth below.

[0054] Both the pyrimidine derivatives and the exogenous multipotentstem cells can be administered by injection with a syringe, insertionwith a catheter or surgical implantation. The pyrimidine derivatives canbe administered at the site of neurological or corporal deficit,systemically (e.g., intravenously), or in the case of neurologicaldeficits of the brain, spinal cord or any tissues accessible by cerebralspinal fluid (CSF), in a brain ventricle. The exogenous multipotent stemcells can be administered at the site of neurological or corporaldeficit, systemically (e.g., intravenously), or in the case ofneurological deficits of the brain, spinal cord or any tissuesaccessible by cerebral spinal fluid (CSF), in a brain ventricle.

[0055] In another in vivo embodiment, the invention provides a methodfor treating an animal with a neurological or corporal deficit. In oneembodiment, the method can comprise administering an effective amount ofa pyrimidine derivative or pharmaceutically acceptable salt thereof suchthat the endogenous stem cell population is stimulated to proliferateand migrate to an area of tissue damage, differentiate in atissue-specific manner and function in a manner that reduces theneurological or corporal deficit. In other embodiments, the inventivemethod further comprises the step of administering multipotent stemcells of App. 1 or App. 2, referenced above, wherein the exogenousmultipotent stem cells are stimulated to proliferate and migrate to anarea of tissue damage, differentiate in a tissue-specific manner andfunction in a manner that reduces the neurological or corporal deficit.Similarly, in related embodiments, the inventive method can comprisesadministering autologous or non-autologous stem cells instead ofadministering the multipotent stem cells of App. 1 or App. 2, whereinthe autologous or non-autologous stem cells are stimulated toproliferate and migrate to an area of tissue damage, differentiate in atissue-specific manner and function in a manner that reduces theneurological or corporal deficit. As an example, tissue-specific stemcells can be isolated from the eventual recipient or another source, andadministered with the pyrimidine derivative. The isolated cells can betreated in vitro with the pyrimidine derivative or be left untreatedwith the pyrimidine derivative. When the autologous or non-autologousstem cells are administered to the human or animal with a neurologicalor corporal deficit, the cells differentiate in a tissue-specific manneraccording to their natural potency. For example, hematopoietic stemcells have some natural, limited capacity to differentiate into certainskin cells. According to this embodiment, hematopoietic stem cells couldbe isolated from the recipient of another source and treated before,concurrently, or after administration to the recipient with a pyrimidinederivative. Such cells are stimulated for proliferation, migration orboth proliferation and migration, and differentiate according to theenvironmental signals they (1) actually encounter and (2) are capable ofnaturally responding to. Thus, hematopoietic stem cells administered toa skin wound with pyrimidine derivative proliferate and migrate due tothe exposure to the pyrimidine derivative and differentiate according tothe environmental signals they encounter in the wound and are capable ofresponding to. Immunosuppressant drugs can be used to suppress anyimmunorejection of non-autologous cells. Similarly, mesenchymal stemcells can be isolated from an animal in need of additional mesenchymalstem cells. Limited numbers of cells can be isolated and treated withpyrimidine derivatives according to the methods of the invention. Suchcells can be stimulated to proliferation, migration or both due toexposure to the pyrimidine derivative. Large numbers of cells can bepropagated in vitro and reintroduced to the donor or other,non-autologous recipient.

[0056] The multipotent stem cells can be administered in the form acluster of two or more cells. The multipotent stem cells can be derivedfrom a tissue or tissue-specific stem cell, for example, a hematopoieticstem cell, a neural stem cell, an epithelial stem cell, an epidermalstem cell, a retinal stem cell, an adipose stem cell and a mesenchymalstem cell, any of which can be obtained from any tissue containing stemcells including but not limited to zygote, blastocyst, embryo, fetus,infant juvenile or adult, and optionally, a human species of any of thepreceding embodiments, whether naturally occurring or engineered.

[0057] When utilizing “more developmentally potent” multipotent stemcells or autologous stem cells or non-autologous stem cells in a clusterof two or more cells, the cluster of multipotent stem cells can compriseless than about 50 percent redifferentiated cells, or more preferablyless than about 25 percent redifferentiated cells, or even morepreferably less than about 10 percent redifferentiated cells, or evenmore preferably less than about 5 percent redifferentiated cells, oreven more preferably less than about 1 percent redifferentiated cells.“Redifferentiated cells” as used herein, refers to cells that haveterminally differentiated during the performance of the methods hereinprior to migration, differentiation and incorporation into host tissueto.

[0058] Similar to other embodiments described above, the multipotentstem cells, optionally in cluster form, are administered by injectingwith a syringe, inserting with a catheter or implanting surgically. Themultipotent stem cells can be administered at the site of neurologicalor corporal deficit, systemically (e.g., intravenously), or in the caseof neurological deficits of the brain, spinal cord or any tissuesaccessible by cerebral spinal fluid (CSF), in a brain ventricle. Inother words, the cells can be implanted to a body cavity that isfluidly-connected to the area of neurological or corporal deficit ordirectly to the area of neurological or corporal deficit. Theneurological deficit is optionally caused by a neurodegenerativedisease, a traumatic injury, a neurotoxic injury, ischemia, adevelopmental disorder, a disorder affecting vision, an injury ordisease of the spinal cord, a demyelinating disease, an autoimmunedisease, an infection, or an inflammatory disease and the corporaldeficit is optionally caused by corporal disease, disorder, injury,trauma, malfunction, degeneration or loss.

[0059] In the methods relating to the in vivo stimulation ofproliferation and migration of endogenous and exogenous mammalian stemcells, an effective amount of pyrimidine derivatives is administered. Aneffective amount can be, for example, a concentration effective toaccomplish aforementioned effects. Non-limiting, exemplaryconcentrations can be between about 0.01 mg/kg/day to 50 mg/kg/day, morepreferably between about 0.1 mg/kg/day to 10 mg/kg/day, even morepreferably between about 1 mg/kg/day to 5 mg/kg/day, and even morepreferably about 3 mg/kg/day. The pyrimidine derivatives can beadministered as necessary to elicit the stimulatory effects, aneffective period, which can be, for example, between about 1 and 60days, or more preferably between about 1 and 30 days, or more preferablybetween about 1 and 15 days, or even more preferably between about 1 and10 days, or more preferably between about 2 and 7 days, or even morepreferably about 5 days.

[0060] The in vivo methods of the invention can further comprise theadministration of a growth factor, including, for example, fibroblastgrowth factor (FGF), epidermal growth factor (EGF) or a combinationthereof.

[0061] The invention also provides methods of stimulating proliferation,migration or both proliferation and migration of mammalian stem cells invitro. In one embodiment, the method comprises the step of contacting amammalian stem cell or in vitro culture thereof with an effective amountof the pyrimidine derivative of formulae (I) or (II) above or apharmaceutically acceptable salt thereof. The stem cell culture can becontacted with the pyrimidine derivative at a concentration effective toproduce the stimulatory effect. For example, a concentration of betweenabout 50 nM to 1 mM can be used, or more preferably between about 500 nMto 500 μM, or even more preferably between about 1 μM to 100 μM, or morepreferably between about 5 μM to 75 μM and even more preferably about 50μM. As with in vivo embodiments, the stem cell culture can be contactedwith pyrimidine derivatives for an effective period, which can be, forexample, between about 1 and 60 days, or more preferably between about 1and 30 days, or more preferably between about 1 and 15 days, or evenmore preferably between about 1 and 10 days, or more preferably betweenabout 2 and 7 days, or even more preferably about 5 days. Also similarto the in vivo embodiments, the cell cultures can be contacted with agrowth factor, for example, FGF, EGF or a combination thereof A growthfactor, as defined herein, refers to a protein, peptide or othermolecule having a growth, proliferative, or trophic effect on the cells(whether “more” or “less” developmentally potent as defined herein) orprogeny thereof. Growth factors used for inducing proliferation includeany trophic factor that allows more or less developmentally potent cellsto proliferate, including any molecule that binds to a receptor on thesurface of the cell to exert a trophic, or growth-inducing effect on thecell. Exemplary proliferation-inducing growth factors include epidermalgrowth factor (EGF), amphiregulin, acidic fibroblast growth factor (aFGFor FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforminggrowth factor alpha (TGFα), and combinations thereof. Preferredproliferation-inducing growth factors include EGF and FGF or acombination thereof. Growth factors are usually added to the culturemedium at concentrations of between about 1 fg/mL to 1 mg/mL.Concentrations between about 1 to 100 ng/mL are usually sufficient.Simple titration experiments routine in the art are used to determinethe optimal concentration of a particular growth factor for a particularcell culture (see, e.g., Cutroneo et al., 2000, Wound Repair Regen, 8:494-502). The method can, in certain embodiments, further comprisecontacting the multipotent stem cell culture with heparin.

[0062] The invention also provides cells that are treated according tothe methods of the invention and are thereby stimulated to proliferate,migrate or both proliferate and migrate in vivo or in vitro. These cellscan be used as an active ingredient in a pharmaceutical composition fortreating a neurological deficit or corporal deficit. In certainembodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier as described below.

[0063] Pharmaceutical compositions optimally comprise a therapeuticallyeffective amount of the stimulated cells of the invention in admixturewith a pharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration. Acceptableformulation materials preferably are nontoxic to the stimulated cellsand the recipients at the dosages and concentrations employed.

[0064] The pharmaceutical compositions of the invention may containformulation materials for modifying, maintaining, or preserving, forexample, pH, osmolarity, viscosity, clarity, color, isotonicity, odor,sterility, stability, rate of dissolution or release, adsorption, orpenetration of the composition, as well as proliferation, migration anddifferentiation capacity of the stimulated cells of the invention.Suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine, or lysine),antimicrobial compounds, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;trimethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R.Gennaro, ed., Mack Publishing Company 1990).

[0065] The primary vehicle or carrier in a pharmaceutical compositionmay be either aqueous or non-aqueous in nature. For example, a suitablevehicle or carrier for injection may be water, physiological salinesolution, or artificial cerebrospinal fluid. Optimal pharmaceuticalcompositions will be determined by a skilled artisan depending upon, forexample, the intended route of administration, delivery format, desireddosage and recipient tissue. See, e.g., REMINGTON'S PHARMACEUTICALSCIENCES, supra. Such compositions may influence the physical state,stability, and effectiveness of the composition.

[0066] Examples of the pharmaceutically acceptable salts of thecompounds of formulae (I) and (II) include hydrochlorides,hydrobromides, sulfates, bisulfites, phosphates, acidic phosphates,acetates, maleates, fumarates, succinates, lactates, tartrates,benzoates, citrates, gluconates, glucanates, methanesulfonates,p-toluenesulfonates and naphthalene-sulfonates which are formed fromacids capable of forming pharmaceutically acceptable anion-containingnontoxic acid addition salts, hydrates thereof, and quaternary ammonium(or amine) salts or hydrates thereof. In a preferred embodiments thepyrimidine derivative of formula (I) is 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H) pyrrolo[²,³-d]pyrimidine maleate (the C₄H₄O₄ maleatesalt), also known as MS-818 (see, for example, Sanyo et al., 1998, J.Neurosci Res. 54: 604-612).

[0067] Thus, the invention advantageously provides methods ofstimulating proliferation and migration of mammalian stem cells in vivoand in vitro, cells produced by those methods, pharmaceuticalcompositions to treat neurological and corporal deficits, and methods ofadministering the cells and pharmaceutical compositions of theinvention.

[0068] Cells can be obtained in any way known in the art and from anytissue, for example, from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue or fromcommercial sources of NSCs (e.g., BioWhittaker, Walkersville, Md.,CC-2599). Tissue from brain can removed using sterile procedures, andthe cells can be dissociated using any method known in the art includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as mincing or treatment witha blunt instrument. Dissociation of neural cells can be carried out intissue culture medium; in a preferred embodiment, the medium fordissociation of juvenile and adult cells is low calcium artificialcerebral spinal fluid (aCSF) having a formula identical to aCSF (124 mMNaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mMD-glucose) except that MgCl₂ is present at a concentration of 3.2 mM andCaCl₂ at a concentration of 0.1 mM. Dissociated cells are centrifuged atlow speed, between 200 and 2000 rpm, usually between 400 and 800 rpm,the suspension medium is aspirated, and the cells are then resuspendedin culture medium. Suspension cultures are preferred if large numbers ofundifferentiated neural stem cell progeny are desired. Cell suspensionsare seeded in any receptacle capable of sustaining cells, preferablyculture plates or roller bottles that inhibit contact-dependent stemcell differentiation, including uncoated flask or a flask that has beentreated to repel the cells.

[0069] While isolation from brain tissue is generally feasible forpreparation of exogenous multipotent cells to be administered with thepyrimidine derivative according to the methods of the instant invention,stem cells from bone marrow (e.g., mesenchymal stem cells) are aparticularly good source of cells for generating multipotent stem cellsof App. 1, because isolation techniques are well established in the arthaving been used for decades in immune disorder bone marrow transplants.Further, the methods of the instant invention can then be carried outwith autologous cells, thus alleviating any concerns over immunologicalrejection. Thus, a patient's own mesenchymal stem cells can be isolated,treated according to the methods of App. 1 and readministered wherenecessary. In contrast, autologous transplants using a neural cellsource, while certainly not impossible, are not as feasible as, forexample, mesenchymal cells.

[0070] Growth of multipotent stem cells under the above cultureconditions induces or permits these cells to form undifferentiated cellclusters. These clusters are optimally grown at a density ofapproximately 50 clusters per T75 flask in 20 mL of the growth mediumconsisting of, for example, DMEM/HAMS F12 (at about 3: 1; Gibco, BRL,Burlington, ON), supplemented with an antibiotic-antimycotic mixture(1:100, penicillin G, streptomycin sulfate, amphotericin B; Gibco), B27(1:50, GIBCO), human recombinant FGF-2 and EGF (20 ng/ml each, R&DSystems, Minneapolis, Minn.) and heparin (5 μg/mL, Sigma, St. Louis,Mo.). The cultures are kept in a CO₂ incubator (about 5% CO₂) at 37° C.To facilitate optimal growth conditions, clusters of two or more cellsare sectioned into quarters approximately every 14 days and fed byreplacing 50% of the medium approximately every 4-5 days. Theseconditions permit rapid and continual growth of MSCs that can beexpanded indefinitely while retaining their multipotent character. Aswith most eukaryotic cells, conditions for culturing should be as closeas possible to physiological conditions. The pH of the culture mediumshould be close to physiological pH, preferably between pH 6-8, morepreferably between about pH 7 to 7.8, with pH 7.4 being most preferred.Physiological temperatures range between about 30° C. to 40° C. Cellsare preferably cultured at temperatures between about 32° C. to about38° C., and more preferably between about 35° C. to about 37° C.Multipotent neural stem cells (MNSCs) prepared and maintained asdisclosed herein continue to exhibit multipotent character after morethan three years of serum-free propagation. Treatment with pyrimidinederivative according to the methods of the invention then transformthese cells into the cells of the invention, cells specially stimulatedfor proliferation, migration or both. If in vitro differentiation isdesired, the cells can be replated in culture dishes in, for example,serum-free basal medium Eagle (BME), which contains Earle's salt andL-glutamine. The cells can be cultured for about 5 days in the absenceof FGF-2, EGF or other extrinsic differentiation factors. When inducedto differentiate in this way, these cultured MNSCs exhibitcharacteristic morphologies of neurons or astrocytes whenimmunohistochemically stained with b-III tubulin (a neuronal cellmarker) or glial fibrillary acidic protein (GFAP, an astrocyte marker).

[0071] MSCs prepared according to the methods of Apps. 1 or 2 andutilized in this invention that are proliferated in serum-free media aregrown in the presence of a substituted deoxyuridine. Examples include ahalo-deoxyuridine such as bromodeoxyuridine (BrdU) or iododeoxyuridine(IrdU), or an alkyl-substituted deoxyuridine such as amethyldeoxyuridine prior to transplantation into a host. The growthmedium used to generate MSCs according to Apps. 1 and 2 for use in thepresent invention comprises the components of the long-term propagationmedia, but also contains an effective amount of substituteddeoxyuridine, for example, concentrations between about 10 nanomolar and100 micromolar, more preferably between about 2 and 50 micromolar, andmore preferably about 10 micromolar bromodeoxyuridine.Pre-transplantation propagation can extend for an effective period, forexample, between about 1 and 10 days, more preferably between about 1and 5 days and more preferably between about 2 and 3 days.

[0072] MSCs prepared according to the methods of Apps. 1 and 2 can beadministered according to the instant invention to an animal withabnormal or degenerative symptoms obtained in any manner, includingthose obtained as a result of age, physical or biological trauma, orneurodegenerative disease and the like, or animal models created by manusing recombinant genetic techniques, such as transgenic and “geneknockout” animals.

[0073] Recipients of the MSCs and pyrimidine derivatives according tothe methods of the invention can be immunosuppressed, either through theuse of immunosuppressive drugs such as cyclosporin, or through localimmunosuppression strategies employing locally appliedimmunosuppressants, but such immunosuppression need not necessarily be aprerequisite in certain immunoprivileged tissues such as, for example,brain and eye tissues or in the case of autologous transplantation. Incertain embodiments, the delivery method of the invention can cause lesslocalized tissue damage to the site of cell damage or malfunction thanexisting methods of delivery.

[0074] MSCs of Apps. 1 and 2 and used herein can be prepared from therecipient's own tissue. In such instances, the progeny of the moredevelopmentally potent cells can be generated from dissociated orisolated tissue and proliferated in vitro using the methods described inApp. 1, App. 2, and herein. In the case of mesenchymal stem cells(MeSCs), progeny can be generated from MeSCs isolated from, for example,bone marrow. Upon suitable expansion of cell numbers, the stem cells ofApps. 1 or 2 can be treated and administered according to the instantinvention into the recipient's affected tissue.

[0075] It is well recognized in the art that transplantation of tissueinto the CNS offers the potential for treatment of neurodegenerativedisorders and CNS damage due to injury. Transplantation of new cellsinto the damaged CNS has the potential to repair damaged circuitries andprovide neurotransmitters thereby restoring neurological function. It isalso recognized in the art that transplantation into other tissue, suchas eye tissue, offers the potential for treatment of degenerativedisorders and tissue damage due to injury. Apps. 1 and 2 provide methodsfor generating more developmentally potent MSCs from lessdevelopmentally potent MSCs. The use of the cells of Apps. 1 or 2, orthe cells of the instant invention specially stimulated forproliferation, migration or both in the treatment of neurologicaldisorders and CNS damage, as well as the use of these MSCs in thetreatment of other tissue damage or degeneration, can be demonstrated bythe use of established animal models known in the art.

[0076] There are significant differences in the method of delivery tothe brain and spinal cord of the cells prepared according to Apps. 1, 2and the present invention, as well as the pyrimidine derivativesdescribed herein, compared to the prior art. One exemplary difference isthat the cells prepared according to Apps. 1, 2 and the presentinvention are transplanted intraventricularly. Further, while thetransplantation of one or more separate more cells of Apps. 1, 2 or theinstant invention is efficacious, such cells are preferably transplantedin the form of clusters of two or more cells via a surgical procedure,injection using a syringe large enough to leave the neurosphere-likeclusters substantially intact, or insertion by catheter. The resultsdisclosed in the examples below indicate that ventricular delivery ofthe cells of Apps. 1 or 2 or the cells of the present invention incluster form can result in migration to the area of damage in the brainand proper neuronal differentiation. Further exemplified below is theeffect of a pyrimidine derivative on the stimulation of proliferationand migration. Another benefit of intraventricular injection is lesstissue destruction, resulting in less localized recruitment of immunecells by the host. This is evidenced by the lack of ventriculardistortion, tumor formation, and increased host astrocyte stainingwithout any immunosuppression.

[0077] The method of delivery of the cells of Apps. 1, 2 or the instantinvention to the brain can be essentially duplicated for otherimmunoprivileged tissue such as, for example, the eye. Delivery ofintact clusters of two or more cells via injection using a syringe largeenough to leave the clusters substantially intact can result inmigration to the area of damage in the eye and proper tissue-specificdifferentiation. Further, administration of pyrimidine derivativesaccording to the methods of the invention can substantially increase theproliferation of endogenous and exogenous MSCs.

[0078] There are examples in the art of intra-tissue injection (brain)of dissociated and partially differentiated NSCs (see, e.g., Benningeret al., 2000, Brain Pathol. 10: 330-341; Blakemore et al., 2000, CellTransplant. 9: 289-294; Rosser et al., 2000, Eur. J. Neurosci. 12:2405-2413; Rubio et al., 2000, Mol. Cell. Neurosci. 16: 1-13). Incontrast, the methods of the instant invention employ injection ofgenerally intact clusters because the dissociation of clusters, in thecase of neural-lineage clusters of cells known as “neurospheres,” cancause immediate senescence and increase the vulnerability of NSCs inculture. See, e.g., Svendsen et al., 1998, J. Neurosci. Methods 85:141-152. As provided by this invention, intraventricular transplantationprovides an alternative route to site-specific injection disclosed inthe prior art. Using intraventricular transplantation, grafted cells cangain access to various structures by the flow of cerebrospinal fluid(CSF), and transplantation of NSCs prepared according to Apps. 1 and 2or the present invention and administered according to the presentinvention in cluster form can act to prevent premature differentiationat inappropriate anatomical sites in the brain and central nervoussystem. Regarding the eye, intraocular administration of clusters ofNSCs prepared according to Apps. 1 and 2 or the present invention, forexample into the vitreous fluid, allows these multipotent cells tomigrate to the area of degeneration or injury and differentiateappropriately.

[0079] Delivery of MSCs of Apps. 1 and 2 and of the present inventioninto other, non-immunoprivileged tissues can also be carried out,particularly when the MSCs are autologous to the recipient.

[0080] Functional integration of the graft into the host's neural tissuecan be assessed by examining the effectiveness of grafts on restoringvarious functions, including but not limited to tests for endocrine,motor, cognitive and sensory functions. Useful motor tests include teststhat quantitate rotational movement away from the degenerated side ofthe brain, and tests that quantitate slowness of movement, balance,coordination, akinesia or lack of movement, rigidity and tremors.Cognitive tests include tests of the ability to perform everyday tasks,as well as various memory tests, including maze performance such as theMorris water maze performance. For example, using the cells and methodsof Apps. 1 and 2, MNSCs injected into the ventricle of 24-month-old ratsafter in vitro expansion displayed extensive and positionalincorporation into the aged host brain with improvement of cognitivescore (FIG. 1), as assessed by the Morris water maze after 4 weeks ofthe transplantation. Results of the experiments disclosed hereinindicate that the aged brain is capable of providing the necessaryenvironment for MSCs of Apps. 1 and 2 and the present invention toretain their multipotent status and demonstrate the potential forneuroreplacement therapies in age associated neurodegenerative disease.

[0081] Functional integration of the graft into the host's other tissuecan be assessed by examining the effectiveness of grafts on restoringvarious functions specific to the injured or degenerated tissue, forexample improvement in sight for transplantation of stem cells of theinvention to the eye. Using the methods of the present invention,substantial stimulation of proliferation of endogenous stem cells can beobserved in the eye with administration of a pyrimidine derivative asdisclosed herein.

[0082] As assessed by the Morris water maze test, improvement in spatialmemory of MSC-transplanted animals (which cells were prepared accordingto Apps. 1 or 2, and apply to the cells of the instant invention) wasaccompanied by incorporation of the MSCs into the brain areas known tobe related to spatial memory. The post-transplant morphology of ratbrain tissue indicates that functional association of the transplantedcells to the host brain occurs. Immunohistochemical analysis revealedthat the bIII-tubulin-positive donor-derived cells found in the cerebralcortex are characterized by having dendrites pointing to the edge of thecortex whereas in the hippocampus, donor-derived neurons exhibitedmorphologies with multiple processes and branches. These differentialmorphologies of the transplanted MSCs in different brain regionsindicate that site-specific differentiation of the MSCs occurs accordingto various factors present in each brain region.

[0083] Strong astrocyte staining was also found in the frontal cortexlayer 3 and CA2 region of hippocampus in rat brains transplanted withMSCs of Apps. 1 and 2, areas where astrocytes are not normally presentin the animal. Migration of the more developmentally potent cells to theCA2 is of particular interest because CA2 pyramidal neurons highlyexpress bFGF, and the expression of bFGF is up-regulated by entorhinalcortex lesions (see, e.g., Eckenstein et al., 1994, Biochem. Pharmacol.47:103-110; Gonzalez et al., 1995, Brain Res. 701: 201-226; Williams etal., 1996, J. Comp. Neurol. 370: 147-158). CA2 pyramidal neurons in thehost brain can express bFGF as a response to a reduction of synaptictransmission, an event that can occur during aging. Subsequently, thisexpressed bFGF can act as a signal for transplanted MSCs of Apps. 1 and2 or the present invention to respond, migrate or proliferate under theinfluence of bFGF produced in the host brain after the transplantation.

[0084] The regions rich in astrocyte staining in transplanted rat brainsare the same regions where extensively stained neuronal fibers wereidentified (FIGS. 2a, 2 d and 2 e). During development, glial cells havemany complex functions, such as neuronal and axonal guidance andproduction of trophic factors (see, e.g., Pundt et al., 1995, Brain Res.695: 25-36). This overlapping distribution of glial and neuronal fibersstrongly suggests that this interaction plays a pivotal role insurvival, migration, and differentiation of the transplanted MSCs.

[0085] Immunohistochemistry of transplanted rat brains reveals asymmetrical distribution of neurons and astrocytes at both sides of thehost brain, indicating that the progeny of the more developmentallypotent cells of Apps. 1, 2 (and those of the present invention) canmigrate. Although astrocytes have been shown to migrate over longdistances following transplantation (see, e.g., Blakemore et al., 1991,Trends Neurosci. 14: 323-327; Hatton et al., 1992, Glia 5: 251-258;Lundberg et al., 1996, Exp. Neurol. 139: 39-53), there is experimentalevidence showing that neurons do not migrate as widely as glial cells(see, e.g., Fricker et al., 1999, J. Neurosci. 19: 5990-6005). Asdisclosed herein, cells derived from the MSCs of Apps. 1 and 2 possesssimilar migratory capacity to astrocyte precursors.

[0086] As MSCs of Apps. 1 and 2 and the present invention can mimicneural stem cells in many regards, relevant information pertaining toneural stem cells is presented, followed by information pertaining tomesenchymal and retinal stem cells. One of skill in the art will readilyrecognize the methods of the invention are not limited to these threetypes of stem cells and instead extend to cover all cell types not yetterminally differentiated.

[0087] Neural-Related

[0088] Due to the generally low proliferation rate of mammalian NSCs,there is a correlation between advancing age and impaired brain functioneven in the absence of specific neurodegenerative disease or physical orbiological brain trauma. Apps. 1 and 2 and the present invention providemethods for counteracting impaired brain function due to advancing agethrough the addition of MSCs (of Apps. 1 and 2 and the presentinvention) capable of proliferation, migration and differentiation inmammalian brain when introduced thereto.

[0089] Physical trauma and biological trauma are additional causes ofimpaired or improper brain function. The term “physical trauma” denotesbrain cell damage due to external sources such as blunt head trauma,severe concussion and the like. Such physical trauma can be localized orgeneral depending on the source and severity of the trauma. The term“biological trauma” denotes any acute brain injury that has its originin a biological process, for example, stroke, aneurysm, epilepsy, braintumor, hypoxia and the like.

[0090] Another source of impaired or improper brain function isneurodegenerative disease. In recent years neurodegenerative disease hasbecome an important concern due to an expanding elderly population thatis at greatest risk for these disorders. Neurodegenerative diseasesinclude, but are not limited Alzheimer's disease, amyotrophic lateralsclerosis (ALS), Parkinson's disease, Pick's disease, Huntington'sdisease, progressive supranuclear palsy, corticobasal degeneration,Parkinson-ALS-dementia complex, Gerstmann-Straussler-Scheinker syndrome,Hallervorden-Spatz disease, Kufs' disease, Wilson's disease, multiplesclerosis (MS), late-onset metachromatic leukodystrophy andadrenoleukodystrophy. The effects of these diseases can be counteractedby administration of the MSCs of Apps. 1 and 2 and the presentinvention.

[0091] There are a variety of organic brain diseases that impair motoror cognitive function. Degeneration in the basal ganglia can lead todiseases with cognitive and motor symptoms, depending on the exactlocation of the degeneration. Motor deficits are a common result ofdegeneration in the basal ganglia. Huntington's chorea is associatedwith the degeneration of neurons in the striatum, which leads toinvoluntary jerking movements in the host. Degeneration of a smallregion called the subthalamic nucleus is associated with violentflinging movements of the extremities in a condition called ballismus,while degeneration in the putamen and globus pallidus is associated witha condition of slow writhing movements or athetosis. In Parkinson'sdisease, degeneration is seen in another area of the basal ganglia, thesubstantia nigra par compacta. This area normally sends dopaminergicconnections to the dorsal striatum, which are important in regulatingmovement. Therapy for Parkinson's disease has centered upon restoringdopaminergic activity to this circuit, which can be accomplished bytransplantation of neural stem cells to this region of the brainaccording to the instant invention

[0092] In Alzheimer's disease, another neurodegenerative disease, thereis substantial cellular degeneration of the forebrain and cerebralcortex. Further, a localized area of the basal ganglia, the nucleusbasalis of Meynert, appears to be selectively degenerated. This nucleusnormally sends cholinergic projections to the cerebral cortex, which arethought to participate in cognitive functions including memory.

[0093] Mesenchymal Related

[0094] Although adult stem cells continue to possess some multipotency,cell types produced from adult stem cells are limited by theirtissue-specific character. For example, human NSCs spontaneouslydifferentiate into brain cells under basal media conditions, but MeSCscannot spontaneously differentiate into neural cells without theaddition of certain factors. These results indicate that each stem cellcontains specific information that would allow it to become a specialtype of cell, i.e., they are partially committed to become a particulartype of cell in a tissue-specific manner. To overcome this barrier ofstem cell lineage, alterations to the cells and their environment arenecessary. However, the exact regulation mechanisms of tissue-specificstem cell fate decisions remain unclear. The absence of this knowledgecreates an important problem, because although MeSCs are rather easy toisolate from bone marrow and to proliferate in culture, they cannotnaturally differentiate into NSCs or other non-mesenchymal-lineagecells. Although the potential therapeutic use of MeSCs in the centralnervous system has been discussed, technologies to induce neural lineagein MeSCs had not been fully established prior to Apps. 1 and 2. Thepresent invention provides methods stimulating proliferation, migrationor both proliferation and migration of the endogenous stem cellpopulation or populations of exogenously introduced cells, such as, forexample, the cells of Apps. 1 or 2.

[0095] MeSCs prepared according to the methods of App. 1 can serve as analternative to NSCs for potential therapeutic use utilizing the methodsof App. 1 and the present invention, which exploit the capacity ofsubstituted deoxyuridine species, such as BrdU, to prime the MeSCs,i.e., remove them from their restricted mesenchymal differentiation pathto the neural stem cell-like (or other lineage, i.e., make them moredevelopmentally potent) differentiation path and pyrimidine derivatives,which stimulate them to proliferate and migrate far above wild-typerates. MeSCs were successfully differentiated into neurons and glia invitro and in vivo using the substituted deoxyuridine pretreatment ofApp. 1. Thus, MeSCs of App. 1 can serve as an alternative to NSCs forpotential therapeutic use in neuroreplacement utilizing the methods ofApp. 1 and the present invention.

[0096] The methods of the instant invention are important in theneuroreplacement field because they enable the expansion of endogenousstem cell numbers in vivo. Further, the methods of the invention areimportant in the neuroreplacement field because they enable thestimulation of proliferation and migration in exogenously introduced,developmentally potent, stem cell populations such as those of Apps. 1or 2. Since the pyrimidine derivative, as used in the instant invention,can be used on various stem cell populations, the invention is not onlyuseful to neuroreplacement but to other kinds of tissue regeneration orreplacement as well.

[0097] Retinal Related

[0098] Retinal degenerative diseases, including macular degeneration,are major causes of blindness. Despite investigations into gene therapy,growth/survival factor injections and vitamin treatments, no effectivevision-restoring treatments are currently available. Visual impairmentcaused by the degeneration of photoreceptors or neural cells has beenconsidered incurable because of a long-held “truism” that neurons do notregenerate during adulthood. However, this statement has been challengedand there is new evidence that these cells do indeed have the potentialto be renewed after maturation, thus opening a door for the developmentof novel therapies to treat visual impairment by retinal regenerationusing stem cell transplantation.

[0099] The capacity for retinal regeneration in cold-blooded vertebrateshas long been recognized. Fish and amphibians continue to make newretinal neurons through a population of retinal stem cells residing atthe peripheral margin of the retina, the so-called “ciliary marginalzone.” Recent studies have provided evidence that birds and adultmammals also possess a zone of cells at the retinal margin analogous tothe ciliary marginal zone of cold-blooded vertebrates. These retinalstem cells are reported not only to generate photoreceptor and otherretinal cells in vitro, but also to differentiate into retinal cellsfollowing transplantation into the retinal area. Although these resultsindicate the possibility of retinal regeneration therapy, an alternativesource of stem cells, or a means to increase the number of endogenousretinal stem cells, is required for clinical applications because thenumber of retinal stem cells is limited.

[0100] Neural stem cells have been isolated from embryonic and adultmammalian brains and have been propagated in vitro in a variety ofculture systems. Using a serum-free unsupplemented media condition, NSCsspontaneously differentiated into bIII-tubulin-, glial fibrillary acidicprotein (GFAP)-, and O4-immunopositive cells, markers' for neurons,astrocytes, and oligodendrocytes, respectively. MSCs treated accordingto methods of Apps. 1 and 2 migrate and differentiate into neurons andglia after transplantation into the brains of 24-month-old rats andsignificantly improved the cognitive functions of these animals. Thisresult suggested that MSCs produced according to Apps. 1 and 2 couldprovide transplantable material to produce a retinal stem cellalternative.

[0101] There are variety of factors involved in the development ofretinal tissue that regulate the proliferation and differentiation ofretinal cells. Transforming growth factor beta 3 (TGF-b3) is thought toregulate cell proliferation during development and also influence thecommitment or the differentiation, or both, of neural progenitor cellsto retinal fates. Treatment of embryonic day-18 rat retinal cultureswith TGF beta-like protein, activin A, causes the progenitor cells inthese cultures to exit the cell cycle and differentiate into rodphotoreceptors, indicating that the TGF family is an important regulatorof photoreceptor differentiation in the developing retina. Treatment ofthe NSCs prepared according to Apps. 1 and 2 can be induced to adopt aretinal differentiation path through exposure to the above factors.Utilizing the methods and reagents of the present invention, bothexogenous MSCs, like those prepared according to Apps. 1 and 2, andendogenous stem cells of the eye can be stimulated to proliferate andmigrate beyond wild-type levels.

[0102] Previous transplantation studies of NSCs into retinal tissue withrd mice (a model of retinitis pigmentosa), mechanical lesions, transientischemia and normal retina have revealed that donor cells migrate intothe retinal area and differentiate into neurons and glia, but they didnot show any retinal cell markers. These results indicated that NSCs arealready committed to become neural tissue, and that this commitment isnot mutable solely by transplantation into the retina. Thus, todifferentiate NSCs (or cells of alternate origin, such as MeSCs) intoretinal cells, alteration of their epigenetic information before retinaltransplantation appeared necessary, something accomplished by themethods of Apps. 1 and 2. Using the methods of Apps. 1 and 2, NSCs oreasily obtainable MeSCs, i.e., can be transformed into MSCs andsubsequently used as alternatives to retinal stem cells to repair oculartissue damage or promote tissue regeneration. Treatment of endogenousmultipotent stem cells populations in vivo or the multipotent stem cellsof Apps. 1 or 2 in vitro according to the methods of the presentinvention, can enhance their number and/or migration and hence increasetheir efficacy in repairing damaged tissue in the eye.

[0103] The inventive methods of Apps. 1 and 2 use BrdU and othersubstituted deoxyuridines to change the cell fate decisions of stemcells. In the case of retinal transplants, these MSCs are treated withTGF-b3 to encourage their commitment change into the various cell typesfound in eye tissue, inter alia, chorid, Buchs and retinal pigmentepithelium cells, rod and cone photoreceptor cells, horizontal cells,bipolar neurons, amacrine, ganglion and optic nerve cells. Thesenon-limiting, exemplary cell types found in eye tissue are collectivelyreferred to as retinal cells. These results are enhanced by the methodsof the present invention wherein the number of MSCs competent to migrateand differentiate appropriately is increased due to stimulatedproliferation.

[0104] There are a variety of neurological and corporal deficits thatcan be addressed using the MSCs of Apps. 1 and 2 and the presentinvention.

[0105] “Neurological Deficits” Amenable to Treatment

[0106] Because the invention relates in part to the discovery thatmultipotent precursor cells can be stimulated to proliferate and migratethrough the brain and other tissues, such MSCs can be used to treatneurological deficits caused by a wide variety of diseases, disorders,and injuries. These insults include, but are not limited to, thefollowing.

[0107] Degenerative Diseases

[0108] Degenerative diseases that can be treated according to themethods of the invention include Alzheimer's disease (AD), Parkinson'sdisease (PD), Huntington's disease (HD), Pick's disease, progressivesupranuclear palsy (PSP), striatonigral degeneration, cortico-basaldegeneration, childhood disintegrative disorder, olivopontocerebellaratrophy (OPCA; including a heritable form), Leigh's disease, infantilenecrotizing encephalomyelopathy, Hunter's disease,mucopolysaccharidosis, various leukodystrophies (such as Krabbe'sdisease, Pelizaeus-Merzbacher disease, and the like), amaurotic(familial) idiocy, Kuf's disease, Spielmayer-Vogt disease, Tay Sachsdisease, Batten disease, Jansky-Bielschowsky disease, Reye's disease,cerebral ataxia, chronic alcoholism, beriberi, Hallervorden-Spatzsyndrome, and cerebellar degeneration.

[0109] Traumatic and Neurotoxic Injuries to the Central Nervous System

[0110] Traumatic and neurotoxic injuries that can be treated accordingto the methods of the invention include gunshot wounds, injuries causedby blunt force, injuries caused by penetration injuries (e.g., stabwounds), injuries caused in the course of a surgical procedure (e.g., toremove a tumor or abscess from the CNS or to treat epilepsy), poisoning(e.g., with MPTP or carbon monoxide), shaken-baby syndrome, adversereactions to medication (including idiosyncratic reactions), drugoverdose (e.g., from amphetamines), and post-traumatic encephalopathy.

[0111] Ischemia

[0112] Any disruption of blood flow or oxygen delivery to the nervoussystem can injure or kill cells, including neurons and glial cells,therein. These injuries can be treated according to the methods of thepresent invention and include injuries caused by a stroke (including aglobal stroke (as may result from cardiac arrest, arrhythmia, ormyocardial infarction) or a focal stroke (as may result from a thrombus,embolus, hemorrhage, or other arterial blockage)), anoxia, hypoxia,partial drowning, myoclonus, severe smoke inhalation, dystonias(including heritable dystonias), and acquired hydrocephalus.

[0113] Developmental Disorders

[0114] Developmental disorders that can be treated according to themethods of the invention include schizophrenia, certain forms of severemental retardation, cerebral palsy (whether caused by infection, anoxia,premature birth, blood type incompatibility: etc. and whether manifestas blindness, deafness, retardation, motor skill deficit, etc.),congenital hydrocephalus, metabolic disorders affecting the CNS, severeautism, Down Syndrome, LHRH/hypothalamic disorder, and spina bifida.

[0115] Disorders Affecting Vision

[0116] Disorders affecting vision, particularly those caused by the lossor failure of retinal cells, can be treated according to the methods andcells of the invention. These disorders include, for example, diabeticretinopathy, serious retinal detachment, retinal damage associated withglaucoma, traumatic injury to the retina, retinal vascular occlusion,macular degeneration (wet or dry), post-surgical healing, tumor,heritable retinal dystrophies, optic nerve atrophy, and other retinaldegenerative diseases. Cells targeted for repair utilizing cells andmethods of the invention include, for example, choroids, Buchs, retinalpigment epithelial (RPE), rods, cones, horizontal cells, bipolarneurons, amacrine, ganglion, and optic nerve.

[0117] Injuries and Diseases of the Spinal Cord

[0118] Injuries to or diseases affecting the spinal cord can also betreated according to the methods of the invention. Such injuries ordiseases include post-polio syndrome, amyotrophic lateral sclerosis,nonspecified spinal degeneration, traumatic injury (such as those causedby automobile or sporting accidents), including any injury that crushes,partially severs, completely severs, or otherwise adversely affects thefunction of cells in the spinal cord), injuries caused by surgery to thespinal cord (e.g., to remove a tumor), anterior horn cell disease, andparalytic diseases.

[0119] Demyelinating or Autoimmune Disorders

[0120] Neurological deficits caused by demyelination or an autoimmuneresponse can be treated according to the methods of the invention. Suchdeficits can be caused by multiple sclerosis, or lupus.

[0121] Infectious or Inflammatory Diseases

[0122] Neurological deficits caused by an infection or inflammatorydisease can be treated according to the methods of the invention.Infections or inflammatory diseases that can cause treatable deficitsinclude Creutzfeldt-Jacob disease and other slow virus infectiousdiseases, AIDS encephalopathy, post-encephalitic Parkinsonism, viralencephalitis, bacterial meningitis and meningitis caused by otherorganisms, phlebitis and thrombophlebitis of intracranial venous sinusessyphilitic Parkinsonism, and tuberculosis of the CNS.

[0123] In addition to the deficits, diseases and disorders set forthexplicitly above, those of ordinary skill in the art are well able torecognize neurological deficits, regardless of their cause, and to applythe methods of the present invention to treat patients who have suchdeficits. In addition to the conditions listed above, that are amenableto treatment with the methods described herein, neurological deficitscan be caused by Lesch-Nyhan syndrome, myasthenia gravis, variousdementias, numerous parasitic diseases, and epilepsy. Further,alleviation of age-related memory loss is an object of the invention.The methods of the invention can be readily applied to alleviateneurological deficits caused by these and other diseases, disorders, orinjuries.

[0124] “Corporal deficits” Amenable to Treatment

[0125] The invention also relates to the amelioration of corporaldeficits utilizing multipotent precursor cells stimulated to divide,migrate through damaged tissue and differentiate in a tissue-specificmanner. Cells according to the invention can be used to treat corporaldeficits caused by a wide variety of diseases, disorders, and injuries,the result of which is trauma, malfunction, degeneration or loss ofmuscle such as, for example, cardiac muscle due to myocardialinfarction. Other examples include malfunction, degeneration or loss ofother cells and tissues apart from those discussed in the neurologicaldeficit section above such as, for example, internal organs. Forexample, liver function can be adversely affected by, among otherthings, disease (e.g., cirrhosis or hepatitis), trauma or age. Otherexemplary internal organs amenable to treatment utilizing theembodiments of the invention include heart, pancreas, kidney, stomach,and lung. Corporal deficits also comprise malfunction, degeneration orloss of skeletal assets such as, for example, vertebrae.

[0126] An advantage of the cells of the invention is that they can begenetically engineered according to routine procedures known in the art(See, e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORYMANUAL. 3rd ed., Cold Spring Harbor Laboratory Press: N.Y.). In certainembodiments, constructs encoding proteins of interest can be provided tothe cells. In other embodiments, constructs that inhibit expression ofundesired proteins can be provided (such as, for example, ribozymes andantisense molecules). In further embodiments, drug resistance genes andmarkers, or detectable markers such as GFP can be provided. Preferably,the marker and other genes are operably and genetically linked to geneexpression regulatory elements (including but not limited to promotersand enhancers) that are operable in a terminally differentiated cellderived from the MNSCs of the invention or in the undifferentiated MNSCsof the invention or both.

[0127] The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the scope of the invention, asdefined by the appended claims.

EXAMPLES Example 1

[0128] Improvement of Cognitive Function in Aged Rat by theTransplantation of NSCs of the Invention

[0129] Human NSCs do not require any exogenous factors fordifferentiation and survived more than three weeks in basal mediawithout the addition of any factor to support their survival (Qu et al.,2001, Neuroreport 12: 1127-32). Thus, it appears that human NSCs producefactors to differentiate and support themselves, which suggested thatthese cells could be transplanted into aged animals after treatmentaccording to the methods of Apps. 1, 2 and the present invention.

[0130] Human NSCs, expanded without differentiation under the influenceof mitogenic factors in supplemented serum-free media and pretreated bythe incorporation of bromodeoxyuridine (BrdU) into the nuclear DNA, wereinjected into the lateral ventricle of mature (6-month-old) and aged(24-month-old) rats. Human NSCs prepared according to the methods of theinvention survived 30 days after xenotransplantation into aged ratbrain, while retaining both multipotency and migratory capacity, andalso improved cognitive function in 24-month-old rats. Cognitivefunction of the animals was assessed by the Morris water maze bothbefore and four weeks after the transplantation of human NSCs of theinvention. Before human NSC transplantation, some aged animals (agedmemory unimpaired animals) cognitively functioned in the range of matureanimals, while others (aged memory impaired animals) functioned entirelyout of the cognitive range of the mature animals. After transplantationof the BrdU-treated human NSCs, most aged animals had cognitive functionin the range of the mature animals. Strikingly, one of the agedmemory-impaired animals showed dramatic improvement in its behavior,functioning even better than the mature animals (FIG. 1a). Statisticalanalysis showed that cognitive function was significantly improved inboth mature and aged memory impaired animals but not in agedmemory-unimpaired animals after BrdU-treated human NSC transplantation(FIG. 1b), which may be due to the physical limitations of the agedanimals. The performance of three of the aged animals deteriorated inthe water maze after transplantation of treated human NSCs. It ispossible that the physical strength of these animals deteriorated duringthe experimental period.

[0131] These behavioral results indicate the beneficial effects of thetransplantation of BrdU-treated human NSCs into the host brain. Afterthe second water maze task, postmortem brains were further analyzed byimmunohistochemistry for human bIII-tubulin and human GFAP, markers forneurons and astrocytes respectively. There was no sign of ventriculardistortion, no evidence of tumor formation, and no strong hostanti-graft immunoreactivity was observed as revealed by weak hostastrocyte staining. Intensely and extensively stained with bIII-tubulin,neurons with BrdU-positive nuclei were found in bilateral singular andparietal cortexes (FIGS. 2a-c) and hippocampus (FIGS. 2d,e). ThebIII-tubulin-positive neurons found in the cerebral cortex were typifiedby a dendrite pointing to the edge of the cortex. In the hippocampus,donor-derived neurons exhibited multiple morphologies, varying incellular size and shape, and one or more processes and branching.

[0132] Generally, GFAP-positive astrocytes were localized near the areawhere neuronal cells were found. On further analysis (overlapping imagesof their distributions), donor-derived astrocytes were found toco-localize with neuronal fibers in the cortex (FIG. 2f). Theseastrocytes were larger than the host glia, with cell bodies 8-10 micronsin diameter and thick processes. Some of these astrocytes had aunilateral morphology (asymmetric), and the immunostaining formed a thinring around the nucleus, while the majority of the processes were formedon the other side. Most cells appeared a symmetrical with processesforming from all sides. The absence of this type of cell in normalanimal without the transplantation of treated human NSCs was confirmedusing immunohistochemistry for rat astrocytes. Host astrocytes had smallcell bodies with multiple delicate processes, and were distributedthroughout the brain mainly in white matter and around the edges of thebrain.

[0133] These results demonstrated that transplanted cells of Apps. 1 and2 migrated in rat brain and differentiated into appropriate cell types.The concomitant improvement in cognitive function indicated thattransplanted MSCs of Apps. 1 and 2 were functionally integrated into therecipient brains.

[0134] The Morris Water Maze:

[0135] The Morris water maze consists of a large circular tank(diameter, 183 cm, wall height, 58 cm), filled with water (27° C.) andopacified by the addition of powdered milk (0.9 kg). Beneath the watersurface (1 cm) near the center of one of the four quadrants of the mazea clear escape platform (height, 34.5 cm) is positioned. The ratsreceive three training trials per day for seven consecutive days, usinga 60 sec inter-trial interval. A training trial consists of placing theanimal in the water for 90 seconds or until the swimming ratsuccessfully locates the platform. If the rat fails to find the platformwithin the 90 seconds, the animal is gently guided to the platform. Forspatial learning assessment, the platform's location remains constant inone quadrant of the maze, but the starting position for each trial isvaried. Every sixth trial is a probe trial, during which the platform isretracted to the bottom of the pool for 30 sec and then raised and madeavailable for escape. The training trials assess the acquisition andday-to-day retention of the spatial task while the probe tests are usedto assess search strategy. At the completion of a spatial learningassessment, one session with six trials of cue training is performedRats are trained to escape to a visible black platform that is raised 2cm above the surface of the water. The location of the platform isvaried from trial to trial to assess sensorimotor and motivationalfunctioning independent of spatial learning ability. Each rat is given30 seconds to reach the platform and is allowed to remain there brieflybefore the 30 second inter-trial interval. Accuracy of performance isassessed using a learning index score computed from the probe trials.The learning index is a derived measure from average proximity(cumulative search error divided by the length of the probe trial) onthe second, third, and fourth interpolated probe trials. Scores fromthese trials are weighted and summed to provide an overall measure ofspatial learning ability. Lower scores on the index indicate a moreaccurate search near the target location; higher scores indicate a morerandom search and poor learning.

[0136] Cell Migration and Differentiation:

[0137] In order to investigate differentiation and/or migration of thecells of Apps. 1 or 2 in the brain, MSCs of those applications weretransplanted into rodent brain. The animals were anesthetized with 50mg/kg pentobarbital (i.p.) and mounted in a stereotaxic apparatus (DavidKopf). Approximately 1×10⁴ to 1×10⁵ cells in 5 μl phosphate-bufferedsaline were injected into the ventricle using a microsyringe attached tothe stereotaxic apparatus. After removing the hair from the surgicalsite using electric razor, an iodine swab was be applied to the area anda 0.5 cm surgical incision was made caudal to rostral in the skin at thesurface of the cranium. The ventricle was stereotaxically localizedusing the following exemplary coordinates: AP=−0.58 mm from bregma,ML=+1 mm, and 2.4 mm below dura (for mouse): AP=−1.4 mm from bregma,ML=+3.3 mm, and 4.5 mm below dura (for rat). A 0.4-mm hole was made inthe cranium by careful drilling. The cells of Apps. 1 or 2 were injectedinto the ventricle using a microsyringe. The injection was deliveredover a period of five minutes and the needle was left in place for anadditional two minutes following the injection. After the injection, thesurgically incised skin was closed by Michel suture clip (2.5×10.75 mm).Ten days post-surgery, proper healing of the incision site was observed,and the Michel sutures were removed.

[0138] The existence and location of the cells of Apps. 1 or 2 afteradministration in rat brain was analyzed as follows. At 30 dayspost-transplantation, the rats were sacrificed by an overdose of sodiumpentobarbital (70 mg/kg, i.p.) and perfused with phosphate bufferedsaline (PBS) followed by 4% paraformaldehyde. Brains were removed andincubated overnight in 4% paraformaldehyde fixative containing 20%sucrose. The brains were sliced into 20 micron coronal sections using acryomicrotome. The sections were washed briefly in PBS and pretreatedwith 1M HCl for 30 minutes at room temperature and neutralized withsodium borate (0.1 M, pH 8.0) for 30 minutes in order to increase theaccessibility of an anti-BrdU antibody to BrdU incorporated in the cellnuclei. After rinsing with PBS, sections were transferred to absolutioncontaining 0.25% Triton X-100 in PBS (PBST) for 30 minutes. The sectionswere then blocked by incubation in PBST containing 3% donkey normalserum for 1 hour, followed by incubating the sections overnight at 48°C. with sheep anti-BrdU (1:1000; Jackson IR Laboratories, Inc. WestGrove, Pa.) or mouse anti-BrdU (1:200; DSHB, Iowa City, Iowa) diluted inPBST. After rinsing the sections in PBS, donkey anti-mouse or donkeyanti-sheep conjugated to rhodamine IgG (Jackson IR Laboratories, Inc.)was added at a 1:200 dilution in PBST and the sections further incubatedfor 2 hours at room temperature in the dark.

[0139] The transplanted cells of Apps. 1 or 2, with BrdU immunopositivenuclei, were stained for human bIII-tubulin and human glial filamentprotein (GFAP). The sections were then washed with PBS and incubatedwith mouse IgG2b monoclonal anti-human bIII-tubulin, clone SDL3D10(1:500, Sigma), goat antihuman GFAP, N-terminal human affinity purified(1:200, Research Diagnostics Inc., Flander, N.J.) or mouse IgG1monoclonal anti-GFAP, clone G-A-5 (1:500, Sigma), respectively,overnight at 48° C. in the dark. After brief washing with PBS to removeexcess primary antibody, the location of primary antibody binding wasthen determined using FITC-conjugated (Jackson IR Laboratories, Inc.)secondary antibody (donkey anti-mouse (1:200) or donkey anti-goat IgG(H+L; 1:200), respectively) by incubating the sections for 2 hours atroom temperature in the dark.

[0140] The sections were then washed with PBS thoroughly before mountingto glass slides. The mounted sections were covered with Vectashieldusing 4′,6-diamidine-2-phenylindole-2HCl (DAPI, Vector Laboratories,Inc., Burlingame, Calif.) for fluorescent microscopic observation.Microscopic images were taken by using an Axiocam digital camera mountedon the Axioscope 2 with Axiovision software (Zeiss).

[0141] NSC Culture:

[0142] NSCs were purchased (BioWhittaker, Walkersville, Md.), andalternatively isolated from human tissue, and cultured in anonsupplemented, serum-free basal medium comprising HAMS-F12 (Gibco,BRL, Burlington, ON); antibiotic-antimycotic mixture (1:100, Gibco); B27(1:50, Gibco); human recombinant FGF-2 and EGF (20 ng/ml each, R and DSystems, Minneapolis, Minn.) and heparin (5 ug/ml, Sigma, St. Louis,Mo.). The cells were incubated at about 37° C. in a 5% CO₂ humidifiedincubation chamber (Fisher, Pittsburgh, Pa.). To facilitate optimalgrowth conditions, NSC clusters were sectioned into quarters every 2weeks and fed by replacing 50% of the medium every 4-5 days. To inhibitdifferentiation, the cells can be propagated on an uncoated flask or aflask that has been treated to repel the cells. To inducedifferentiation, these cells can be replated in the culture dishes(about 1×10⁵ per dish) in the serum-free basal medium Eagle (BME), whichcomprises Earle's salt and L-glutamine, and cultured for about 5 days inthe absence of FGF-2 and EGF and without the addition of other extrinsicdifferentiation factors. NSCs cultured in this serum-free medium canspontaneously undergo differentiation into neuronal cell types.

Example 2

[0143] Increase of Endogenous Stem Cell Proliferation by a PyrimidineDerivative

[0144] To investigate the effect of MS-818, a pyrimidine derivative, onstem cell population in vivo, MS-818 (3 mg/kg/day, i.p.) was injectedfor 5 days into aged (27-month old) male Fisher 344 rats. The samevolume of saline was injected into control animals. Bromodeoxyuridine.(BrdU) (100 mg/kg/day i.p.) was then injected for 3 days. Twenty-fourhours after the last injection, the brains were removed and fixed forimmunohistochemical detection of the proliferating cells byimmunostaining for BrdU. The number of BrdU positive cells increasedmore than seven fold in the cerebral cortices of MS-818-treated animalscompared to those of controls (FIGS. 3a,b,e), indicating an increasedneural stem cell population in the brain. In the area of thesubventricular zone, a significant increase not only in theproliferation but also in the migration of stem cells was found (FIGS.3c,d). When this compound was injected directly into the vitreous cavity(10 μg one time injection), a dramatic increase in the number ofBrdU-positive cells was found in the retinal ciliary marginal zone (FIG.4) after three days.

[0145] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

We claim:
 1. A method of stimulating proliferation, migration or bothproliferation and migration of endogenous mammalian stem cells in vivo,the method comprising introducing an effective amount of a pyrimidinederivative of formula (1) or (2), or a pharmaceutically acceptable saltthereof, for an effective period to a mammal,

wherein R1 to R8 independently represent a hydrogen atom, a lower alkylgroup, CH₃OCH₂CH₂—, CH₂CONH₂, —COCH₃, —COC₂H₅ or —CH₂OCOC₂H₅, and Xrepresents ═NH, ═N—CH₃, ═N—C₂H₅, ═N-ph, ═N—COOC₂H₅, ═N—SO₂CH₃, ═CH₂,═CHCH₃, ═CHC₂H₅, —O— or —S— in which ph stands for a phenyl group. 2.The method of claim 1, wherein the method stimulates proliferation ofendogenous mammalian stem cells in vivo.
 3. The method of claim 1,wherein the method stimulates migration of endogenous mammalian stemcells in vivo.
 4. The method of claim 1, wherein the method stimulatesproliferation and migration of endogenous mammalian stem cells in vivo.5. A method of stimulating proliferation, migration or bothproliferation and migration of exogenous mammalian stem cells in vivo,the method comprising introducing an effective amount of a pyrimidinederivative of formula (1) or (2), or a pharmaceutically acceptable saltthereof, for an effective period to a mammal that has had moredevelopmentally potent cells administered thereto

wherein R1 to R8 independently represent a hydrogen atom, a lower alkylgroup, CH₃OCH₂CH₂—, CH₂CONH₂, —COCH₃, —COC₂H₅ or —CH₂OCOC₂H₅, and Xrepresents ═NH, ═N—CH₃, ═N—C₂H₅, ═N-ph, ═N—COOC₂H₅, ═N—SO₂CH₃, ═CH₂,═CHCH₃, ═CHC₂H₅, —O— or —S— in which ph stands for a phenyl group. 6.The method of claim 5, wherein the method stimulates proliferation ofexogenous mammalian stem cells in vivo.
 7. The method of claim 5,wherein the method stimulates migration of exogenous mammalian stemcells in vivo.
 8. The method of claim 5, wherein the method stimulatesproliferation and migration of exogenous mammalian stem cells in vivo.9. A method of stimulating proliferation, migration or bothproliferation and migration of mammalian stem cells in vitro, the methodcomprising contacting a mammalian stem cell with an effective amount ofa pyrimidine derivative of formula (1) or (2), or a pharmaceuticallyacceptable salt thereof, for an effective period,

wherein R1 to R8 independently represent a hydrogen atom, a lower alkylgroup, CH₃OCH₂CH₂—, CH₂CONH₂, —COCH₃, —COC₂H₅ or —CH₂OCOC₂H₅, and Xrepresents ═NH, ═N-CH₃, ═N-C₂H₅, ═N-ph, ═N—COOC₂H₅, ═N—SO₂CH₃, ═CH₂,═CHCH, ═CHC₂H₅, —O— or —S— in which ph stands for a phenyl group. 10.The method of claim 9, wherein the method stimulates proliferation ofmammalian stem cells in vitro.
 11. The method of claim 9, wherein themethod stimulates migration of mammalian stem cells in vitro.
 12. Themethod of claim 9, wherein the method stimulates proliferation andmigration of mammalian stem cells in vitro.
 13. A method for treating ananimal having a neurological or corporal deficit, the method comprisingthe step of: (a) administering a pyrimidine derivative of formula (1) or(2), or a pharmaceutically acceptable salt thereof to the patient;wherein the endogenous stem cell population is stimulated to proliferateand migrate to an area of tissue damage, differentiate in atissue-specific manner and function in a manner that reduces theneurological or corporal deficit.
 14. A method for treating an animalhaving a neurological or corporal deficit, the method comprising thestep of: (a) administering a pyrimidine derivative of formula (1) or(2), or a pharmaceutically acceptable salt thereof to the patient; and(b) administering more developmentally potent cells; wherein the moredevelopmentally potent cells are stimulated to proliferate and migrateto an area of tissue damage, differentiate in a tissue-specific mannerand function in a manner that reduces the neurological or corporaldeficit.
 15. A method for treating an animal having a neurological orcorporal deficit, the method comprising the step of: (a) administering apyrimidine derivative of formula (1) or (2), or a pharmaceuticallyacceptable salt thereof to the patient; and (b) administering autologousstem cells; wherein the autologous stem cells are stimulated toproliferate and migrate to an area of tissue damage, differentiate in atissue-specific manner and function in a manner that reduces theneurological or corporal deficit.
 16. A method for treating an animalhaving a neurological or corporal deficit, the method comprising thestep of: (a) administering a pyrimidine derivative of formula (1) or(2), or a pharmaceutically acceptable salt thereof to the patient; and(b) administering non-autologous stem cells; wherein the non-autologousstem cells are stimulated to proliferate and migrate to an area oftissue damage, differentiate in a tissue-specific manner and function ina manner that reduces the neurological or corporal deficit.
 17. Themethod of any one of claims 1 through 16 wherein the pyrimidinederivative is MS-818.
 18. The method of claim 14, 15 or 16 wherein themore developmentally potent cells or autologous stem cells ornon-autologous stem cells form a cluster of two or more cells.
 19. Themethod of claim 14, 15 or 16 wherein the more developmentally potentcells or autologous stem cells or non-autologous stem cells are derivedfrom a tissue or tissue-specific stem cell.
 20. The method of claims 9,15, 16 or 19 wherein the stem cell is a hematopoietic stem cell, aneural stem cell, an epithelial stem cell, an epidermal stem cell, aretinal stem cell, an adipose stem cell or a mesenchymal stem cell. 21.The method of claims 9, 15, 16 or 19 wherein the stem cell is amesenchymal stem cell.
 22. The method of claims 9, 15, 16 or 19 whereinthe stem cell is obtained from a zygote, blastocyst, embryo, fetus,infant juvenile or adult.
 23. The method of claims 9, 15, 16 or 19wherein the stem cell is obtained from a human.
 24. The method of claim8 wherein the cluster of two or more of the more developmentally potentcells or autologous stem cells or non-autologous stem cells compriseless than about 50 percent redifferentiated cells.
 25. The method ofclaim 18 wherein the cluster of two or more of the more developmentallypotent cells or autologous stem cells or non-autologous stem cellscomprise less than about 25 percent redifferentiated cells.
 26. Themethod of claim 18 wherein the cluster of two or more of the moredevelopmentally potent cells or autologous stem cells or non-autologousstem cells comprise less than about 10 percent redifferentiated cells.27. The method of claim 18 wherein the cluster of two or more of themore developmentally potent cells or autologous stem cells ornon-autologous stem cells comprise less than about 5 percentredifferentiated cells.
 28. The method of claim 18 wherein the clusterof two or more of the more developmentally potent cells or autologousstem cells or non-autologous stem cells comprise less than about 1percent redifferentiated cells.
 29. The method of claim 1, 2, 3, 4, 5,6, 7 or 8 further comprising administering a growth factor.
 30. Themethod of claim 29 wherein the growth factor is fibroblast growthfactor, epidermal growth factor or a combination thereof.
 31. The methodof claim 29 wherein the growth factor is a combination of fibroblastgrowth factor and epidermal growth factor.
 32. The method of claim 29wherein the growth factor is fibroblast growth factor.
 33. The method ofclaim 29 wherein the growth factor is epidermal growth factor.
 34. Themethod of claim 9 further comprising the step of administering a growthfactor.
 35. The method of claim 34 wherein the growth factor isfibroblast growth factor, epidermal growth factor or a combinationthereof.
 36. The method of claim 34 where in the growth factor is acombination of fibroblast growth factor and epidermal growth factor. 37.The method of claim 34 wherein the growth factor is fibroblast growthfactor.
 38. The method of claim 34 wherein the growth factor isepidermal growth factor.
 39. The method of claim 9 further comprisingthe step of contacting the mammalian stem cell with heparin.
 40. Cellsstimulated for proliferation, migration or both proliferation andmigration according to the methods of claim 9 or
 34. 41. Apharmaceutical composition for treating a neurological deficit orcorporal deficit comprising, as an active ingredient, the cellsaccording to claim
 40. 42. The pharmaceutical composition according toclaim 41 further comprising a pharmaceutically acceptable carrier. 43.The method of claim 14, 15 or 16 wherein the more developmentally potentcells or the autologous stem cells or the non-autologous stem cells areadministered by injecting said cells with a syringe, inserting the moredevelopmentally potent cells with a catheter or surgically implantingthe more developmentally potent cells.
 44. The method of claim 43wherein the more developmentally potent cells or the autologous stemcells or the non-autologous stem cells comprise a cluster of two or morecells.
 45. The method of claim 43 wherein the more developmentallypotent cells or the autologous stem cells or the non-autologous stemcells are injected with a syringe into a body cavity that isfluidly-connected to the area of neurological or corporal deficit. 46.The method of claim 43 wherein the more developmentally potent cells orthe autologous stem cells or the non-autologous stem cells are insertedwith a catheter into a body cavity that is fluidly-connected to the areaof neurological or corporal deficit.
 47. The method of claim 43 whereinthe more developmentally potent cells or the autologous stem cells orthe non-autologous stem cells are surgically implanted into a bodycavity that is fluidly-connected to the area of neurological or corporaldeficit.
 48. The method of claim 43 wherein the more developmentallypotent cells or the autologous stem cells or the non-autologous stemcells are injected with a syringe to the area of neurological orcorporal deficit.
 49. The method of claim 43 wherein the moredevelopmentally potent cells or the autologous stem cells or thenon-autologous stem cells are injected with a catheter to the area ofneurological or corporal deficit.
 50. The method of claim 43 wherein themore developmentally potent cells or the autologous stem cells or thenon-autologous stem cells are surgically implanted to the area ofneurological or corporal deficit.
 51. The method of claim 43, whereinthe neurological deficit is caused by a neurodegenerative disease, atraumatic injury, a neurotoxic injury, ischemia, a developmentaldisorder, a disorder affecting vision, an injury or disease of thespinal cord, a demyelinating disease, an autoimmune disease, aninfection, or an inflammatory disease.
 52. The method of claim 43,wherein the corporal deficit is caused by corporal disease, disorder,injury, trauma, malfunction, degeneration or loss.